Carbamoyl-phosphate synthetase gene of coryneform bacteria and method for producing L-arginine

ABSTRACT

A DNA fragment which encodes a polypeptide defined in the following (a) or (b), and a polypeptide defined in the following (c) or (d):  
     (a) a polypeptide which has at least the amino acid sequence of the amino acid numbers 50 to 393 in SEQ ID NO: 2 shown in Sequence Listing,  
     (b) a polypeptide which has at least the amino acid sequence of the amino acid numbers 50 to 393 in SEQ ID NO: 2 shown in Sequence Listing including substitution, deletion, insertion, addition, or inversion of one or several amino acids, and can constitute a protein having a carbamoyl-phosphate synthetase activity with a large subunit of carbamoyl-phosphate synthetase having the amino acid sequence of SEQ ID NO: 3,  
     (c) a polypeptide which has the amino acid sequence of SEQ ID NO: 3 shown in Sequence Listing,  
     (d) a polypeptide which has the amino acid sequence of SEQ ID NO: 3 shown in Sequence Listing including substitution, deletion, insertion, addition, or inversion of one or several amino acids, and can constitute a protein having a carbamoyl-phosphate synthetase activity with a small subunit of carbamoyl-phosphate synthetase having the amino acid sequence of the amino acid numbers 50 to 393 in SEQ ID NO: 2.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to carbamoyl-phosphate synthetaseof coryneform bacteria, and a gene therefor. The gene can be utilizedfor production of carbamoyl-phosphate synthetase and subunits thereof,breeding of L-arginine-producing bacteria and nucleic acid-producingbacteria and so forth.

[0003] 2. Description of the Related Art

[0004] Carbamoyl-phosphate synthetase is an enzyme that catalyzes thereactions producing carbamoyl phosphate from carbonic acid, ATP andglutamine. Carbamoyl phosphate produced by these reactions serves as asource of carbamoyl group required for the reaction producing citrullinefrom ornithine in the L-arginine biosynthetic pathway. Furthermore,carbamoyl aspartate produced from aspartic acid and carbamoyl phosphateis one of the intermediates of the pyrimidine biosynthesis systemincluding uridine 5′-monophosphate.

[0005] Carbamoyl-phosphate synthetase consists of two subunits, and ithas been known for bacteria belonging to the genus Escherichia orBacillus that those subunits are encoded by carA and carB genes.

[0006] However, as for coryneform bacteria, there have been no findingsabout the carbamoyl-phosphate synthetase activity and enzymes therefor,and any genes therefor have not been elucidated.

[0007] Incidentally, it has been reported that when a transformant ofEscherichia coli to which introduced a plasmid harboring the genes carA,carB, argI and arg box was cultured in the medium added with glutaminewhich is substrate of carbamoyl-phosphate synthetase, the concentrationof intracellular L-arginine was the same as that of a control strain towhich only the vector was introduced. However, when the transformant wascultured in a medium added with glutamine accompanied with ornithinewhich is a substrate of ArgI together with carbamoyl phosphate, theconcentration of intracellular L-arginine was higher than that of thecontrol strain (Malamy M. et al., Applied Environmental Microbiology,63(1), 33 (1997)). From these result, it was suggested that therate-determining step of synthesis of L-arginine is supply of ornithine.

[0008] There was thought to be a possibility that the rate-determiningstep of supply of ornithine is N-acetylglutamine synthetase (ArgA). ArgAsuffers feedback inhibition by the final product, L-arginine, in thebiosynthesis pathway of Escherichia coli.

[0009] As for the strain in which argA gene coding for feedbackinhibition-desensitized ArgA was amplified by plasmid, the concentrationof intracellular L-arginine was increased even in a medium added withonly glutamine as well as in a medium added with both glutamine-andornithine. However, farther increase of concentration of intracellularL-arginine was not observed in the case that the strain was culturedwith addition of glutamine, or glutamine and ornithin, also in the casethat the both of carA and carB genes were further amplified in thestrain (Malamy M. et al., Applied Environmental Microbiology, 64(5),1805 (1998)).

[0010] On the other hand, any attempts have not been reported to enhanceL-arginine productibity of microorganisms by utilizing a gene coding forcarbamoyl-phosphate synthetase derived from coryneform bacterium.

SUMMARY OF THE INVENTION

[0011] An object of the present invention is to providecarbamoyl-phosphate synthetase of coryneform bacteria, a gene coding forit, and a method for producing L-arginine with a microorganism utilizingthe gene.

[0012] The inventors of the present invention eagerly studied in orderto achieve the aforementioned object. As a result, the inventorssuccessfully obtained a DNA fragment containing the carA gene and thecarB gene from a wild strain of Brevibacterium lactofermentum byutilizing a carB-deficient strain of Escherichia coli, and thusaccomplished the present invention.

[0013] That is, the present invention provides the followings.

[0014] (1) A DNA fragment which encodes a polypeptide defined in thefollowing (A) or (B):

[0015] (A) a polypeptide which has an amino acid sequence comprises atleast the amino acid numbers 50 to 393 of the amino acid sequence of SEQID NO: 2,

[0016] (B) a polypeptide which has an amino acid sequence comprises atleast the amino acid numbers 50 to 393 of the amino acid sequence of SEQID NO: 2 including substitution, deletion, insertion, addition, orinversion of one or several amino acids, and can constitute a proteinhaving a carbamoyl-phosphate synthetase activity with a large subunit ofcarbamoyl-phosphate synthetase comprising the amino acid sequence of SEQID NO: 3.

[0017] (2) A DNA fragment which encodes a polypeptide defined in thefollowing (C) or (D):

[0018] (C) a polypeptide which comprises the amino acid sequence of SEQID NO: 3,

[0019] (D) a polypeptide which comprises the amino acid sequence of SEQID NO: 3 including substitution, deletion, insertion, addition, orinversion of one or several amino acids, and can constitute a proteinhaving a carbamoyl-phosphate synthetase activity with a small subunit ofcarbamoyl-phosphate synthetase having an amino acid sequence comprisesat least the amino acid numbers 50 to 393 of the amino acid sequence ofSEQ ID NO: 2.

[0020] (3) A DNA fragment encoding a polypeptide which comprises theamino acid sequence of SEQ ID NO: 3 including substitution, deletion,insertion, addition, or inversion of one or several amino acids, and canconstitute a protein having a carbamoyl-phosphate synthetase activity.

[0021] (4) A DNA fragment which encodes a polypeptide defined in thefollowing (a) or (b), and a polypeptide defined in the following (c) or(d):

[0022] (a) a polypeptide which has an amino acid sequence comprising atleast the amino acid numbers 50 to 393 in SEQ ID NO: 2,

[0023] (b) a polypeptide which has an amino acid sequence comprising atleast the amino acid numbers 50 to 393 in SEQ ID NO: 2 includingsubstitution, deletion, insertion, addition, or inversion of one orseveral amino acids, and can constitute a protein having acarbamoyl-phosphate synthetase activity with a large subunit ofcarbamoyl-phosphate synthetase comprising the amino acid sequence of SEQID NO: 3,

[0024] (c) a polypeptide which comprises the amino acid sequence of SEQID NO: 3,

[0025] (d) a polypeptide which comprises the amino acid sequence of SEQID NO: 3 including substitution, deletion, insertion, addition, orinversion of one or several amino acids, and can constitute a proteinhaving a carbamoyl-phosphate synthetase activity with a small subunit ofcarbamoyl-phosphate synthetase having an amino acid sequence comprisingthe amino acid numbers 50 to 393 in SEQ ID NO: 2.

[0026] (5) The DNA fragment according to (1), which has a nucleotidesequence comprising at least the nucleotide numbers 430 to 1461 in thenucleotide sequence of SEQ ID NO: 1.

[0027] (6) The DNA fragment according to (2), which has a nucleotidesequence comprising at least the nucleotide numbers 1756 to 4809 in thenucleotide sequence of SEQ ID NO: 1.

[0028] (7) The DNA fragment according to (3), which has a nucleotidesequence comprising at least the nucleotide numbers 430 to 4809 in thenucleotide sequence of SEQ ID NO: 1.

[0029] (8) A protein which comprises a polypeptide defined in thefollowing (a) or (b), and a polypeptide defined in the following (c) or(d):

[0030] (a) a polypeptide which has an amino acid sequence comprising atleast the amino acid numbers 50 to 393 in SEQ ID NO: 2,

[0031] (b) a polypeptide which has an amino acid sequence comprising atleast the amino acid numbers 50 to 393 in SEQ ID NO: 2 includingsubstitution, deletion, insertion, addition, or inversion of one orseveral amino acids, and can constitute a protein having acarbamoyl-phosphate synthetase activity with a large subunit ofcarbamoyl-phosphate synthetase comprising the amino acid sequence of SEQID NO: 3,

[0032] (c) a polypeptide which comprises the amino acid sequence of SEQID NO: 3,

[0033] (d) a polypeptide which comprises the amino acid sequence of SEQID NO: 3 including substitution, deletion, insertion, addition, orinversion of one or several amino acids, and can constitute a proteinhaving a carbamoyl-phosphate synthetase activity with a small subunit ofcarbamoyl-phosphate synthetase having an amino acid sequence comprisingat least the amino acid numbers 50 to 393 in SEQ ID NO: 2.

[0034] (9) A coryneform bacterium which is transformed with a DNAfragment according to any one of (1) to (7).

[0035] (10) A microorganism which has enhanced intracellularcarbamoyl-phosphate synthetase activity, and has L-arginineproductivity.

[0036] (11) The microorganism according to (10), wherein the enhancedintracellular carbamoyl-phosphate synthetase activity is obtained byincreasing copy number of DNA encoding carbamoyl-phosphate synthetase ofthe microorganism, or by modifying an expression regulation sequence sothat expression of the gene encoding carbamoyl-phosphate synthetase inthe cell should be enhanced.

[0037] (12) The microorganism according to (11), wherein the DNA is aDNA fragment according to any one of (1) to (7).

[0038] (13) The microorganism according to (12), which is a coryneformbacterium.

[0039] (14) A method for producing of L-arginine, comprising the stepsof culturing a coryneform bacterium according to any one of (10) to (13)in a medium to produce and accumulate L-arginine in the medium, andcollecting the L-arginine from the medium.

[0040] The present invention provides genes coding for the subunits thatconstitute carbamoyl-phosphate synthetase. The gene can be utilized forproduction of carbamoyl-phosphate synthetase and subunits thereof,breeding of L-arginine-producing bacteria and nucleic acid-producingbacteria and so forth. Aditionally, L-arginine can be producedefficiently according to the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0041]FIG. 1 shows the structure of plasmid pl9 containing the carA geneand carB gene.

[0042]FIG. 2 shows a construction process of plasmid pK1.

[0043]FIG. 3 shows a construction process of plasmid pSFK6.

DETAIL DESCRIPTION OF THE INVENTION

[0044] Hereafter, the present invention will be explained in detail.

[0045] <1> DNA of the Present Invention

[0046] The DNA of the present invention can be obtained from achromosome DNA library of coryneform bacteria prepared with vectors suchas plasmids by selection of the DNA using a microorganism which isdeficient in carA or carB, for example, Escherichia coli RC50 (carA50,tsx⁻273, λ⁻, rpsL135 (str^(R)), malt1 (λR), xylA7, thi⁻¹ ; Mol. Gen.Genet., 133, 299 (1974)), Escherichia coli JEF8 (thr⁻31, ΔcarB, relA⁻,metB1, Mol. Gen. Genet., 133, 299 (1974)) and so forth. Because amicroorganism which is deficient in carA or carB exhibits L-arginine anduracil auxotrophy, a DNA fragment can be obtained by transforming such amicroorganism with a chromosome DNA library, selecting clones in whichthe auxotrophy is complemented, and recovering a recombinant vector fromthe selected transformants.

[0047] The coryneform bacteria used for preparing a chromosome DNAlibrary are not particularly limited, and examples thereof includebacteria having been hitherto classified into the genus Brevibacteriumbut united into the genus Corynebacterium at present (Int. J. Syst.Bacteriol., 41, 255 (1981)), and include bacteria belonging to the genusBrevibacterium closely relative to the genus Corynebacterium, morespecifically, wild strains of Brevibacterium lactofermentum and soforth. Chromosome DNA of coryneform bacteria can be prepared by, forexample, the method of Saito and Miura (Biochem. Biophys. Acta., 72,619, (1963)), the method of K. S. Kirby (Biochem. J., 64, 405, (1956))and so forth.

[0048] A chromosome DNA library can be obtained by partially digestingchromosome DNA with suitable restriction enzymes, ligating each of theobtained DNA fragments to a vector DNA autonomously replicable inEscherichia coli cells to prepare a recombinant DNA, and introducing theDNA into Escherichia coli. The vector is not particularly limited solong as it is a vector usually used for genetic cloning, and plasmidvectors such as pUC19, pUC18, pUC118, and pUC119, phage vectors such asλ phage DNA and so forth can be used. Further, a vector autonomouslyreplicable in both of Escherichia coli cells and coryneform bacteriumcells may also be used. Such a vector can be constructed by ligating avector for Escherichia coli and pAM330, which is a cryptic plasmid ofBrevibacterium lactofermentum (see Japanese Patent Laid-open No.58-67699).

[0049] Specific examples of the vector autonomously replicable withinboth of Escherichia coli and coryneform bacterium cells include pSAC4(see the examples mentioned below), pHK4 (see Japanese Patent Laid-openNo. 5-7491) and so forth. Escherichia coli HB101 harboring pHK4 wasdesignated as Escherichia coli AJ13136, and it was deposited at theNational Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology, Ministry of International Trade andIndustry (postal code 305-8566, 1-3 Higashi 1-chome, Tsukuba-shi,Ibaraki-ken, Japan) on Aug. 1, 1995, and received an accession number ofFERM BP-5186.

[0050] The transformation of Escherichia coli cells can be performed by,for example, the method of D. A. Morrison (Methods in Enzymology, 68,326, 1979.), the method of treating recipient cells with calciumchloride so as to increase the permeability of DNA (Mandel, M. and Higa,A., J. Mol. Biol., 53, 159 (1970)) and so forth. As for methods forpreparation of chromosome DNA library, preparation of plasmid DNA, anddigestion and ligation of DNA, as well as methods for PCR, preparationof oligonucleotides and hybridization mentioned hereinafter,conventional methods well known to those skilled in the art can be used.Such methods are described in Sambrook, J., Fritsch, E. F. and Maniatis,T., “Molecular Cloning, A Laboratory Manual, Second Edition”, ColdSpring Harbor Laboratory Press, (1989) and so forth.

[0051] A nucleotide sequence of a DNA fragment containing carA and carBobtained as described above is represented as SEQ ID NO: 1 in SequenceListing. This sequence contains two open reading frames (ORF, nucleotidenumbers 283 to 1461 and nucleotide numbers 1756 to 4809). The upstreamORF is carA, and the downstream ORF is carB. The amino acid sequencesencoded by these ORFs are shown in SEQ ID NOS: 2 and 3, respectively.According to the present invention, a peptide encoded by carA isreferred to as a small subunit, and a peptide encoded by carB isreferred to as a large subunit. As for the coding region of carA, GTG ofthe nucleotide numbers 283 to 285 is indicated as the initiation codonin Sequence Listing. However, GTG of the nucleotide numbers 415 to 417or ATG of the nucleotide numbers 430 to 432 may possibly be theinitiation codon. In any case, an active small subunit can be obtainedby using a longer open reading frame for the upstream region for theexpression of carA. The amino acid corresponding to the GTG as theinitiation codon is indicated as valine for each subunit, but it may bemethionine, valine or formylmethionine.

[0052] The small subunit of the carbamoyl-phosphate synthetase of thepresent invention is, for example, a polypeptide having the amino acidsequence of the amino acid numbers 50 to 393 in SEQ ID NO: 2,polypeptide having the amino acid sequence of the amino acid numbers 45to 393 in SEQ ID NO: 2, polypeptide having the amino acid sequence ofthe amino acid numbers 1 to 393 in SEQ ID NO: 2 or the like. The largesubunit of the carbamoyl-phosphate synthetase of the present inventionis, for example, a polypeptide having the amino acid sequence shown asSEQ ID NO: 3.

[0053] According to the present invention, the DNA coding for the smallsubunit may be one coding for an amino acid sequence which contains theamino acid sequence of the amino acid numbers 50 to 393 in SEQ ID NO: 2including substitution, deletion, insertion, addition, or inversion ofone or several amino acids, or one coding for a polypeptide which canconstitute a protein having a carbamoyl-phosphate synthetase activitywith the large subunit.

[0054] According to the present invention, the DNA coding for the largesubunit may be one coding for an amino acid sequence which contains theamino acid sequence of SEQ ID NO: 3 including substitution, deletion,insertion, addition, or inversion of one or several amino acids, or onecoding for a polypeptide which can constitute a protein having acarbamoyl-phosphate synthetase activity with the small subunit.Alternatively, it may be one coding for a protein which has the aminoacid sequence of SEQ ID NO: 3 including substitution, deletion,insertion, addition, or inversion of one or several amino acids, and hasa carbamoyl-phosphate synthetase activity.

[0055] Furthermore, a DNA that encodes carbamoyl-phosphate synthetasecontaining a mutation or mutations in the small subunit or the largesubunit, or both of them also falls within the scope of the DNA of thepresent invention.

[0056] The term “several amino acids” preferably means 1 to 20 aminoacids, more preferably 1 to 10 amino acids.

[0057] DNA, which encodes the substantially same peptide as the smallsubunit or the large subunit as described above, is obtained, forexample, by modifying the nucleotide sequence of the DNA encoding thesmall subunit or the large subunit, for example, by means of thesite-directed mutagenesis method so that one or more amino acid residuesat a specified site of the gene involve substitution, deletion,insertion, addition, or inversion. DNA modified as described above maybe obtained by the conventionally known mutation treatment. The mutationtreatment includes a method for treating DNA coding for the smallsubunit or the large subunit in vitro, for example, with hydroxylamine,and a method for treating a microorganism, for example, a bacteriumbelonging to the genus Escherichia harboring DNA coding for the smallsubunit and the large subunit with ultraviolet irradiation or a mutatingagent such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and nitrousacid usually used for the mutation treatment.

[0058] The substitution, deletion, insertion, addition, or inversion ofnucleotide as described above also includes mutation (mutant or variant)which naturally occurs, for example, the difference in strains, speciesor genera of the microorganism having the small subunit and/or the largesubunit.

[0059] The DNA, which encodes substantially the same protein ascarbamoyl-phosphate synthetase, is obtained by expressing DNA havingmutation as described above in an appropriate cell, and investigatingthe carbamoyl-phosphate synthetase activity of an expressed product. Thecarbamoyl-phosphate synthetase activity can be measured by the knownmethod (Journal of Genral Microbiology, 136, 1177-1183 (1990)). The DNA,which encodes substantially the same protein as carbamoyl-phosphatesynthetase, is also obtained by isolating DNA which is hybridizable withDNA having, for example, a nucleotide sequence corresponding tonucleotide numbers of 283 to 1461 or 1756 to 4809 of the nucleotidesequence of SEQ ID NO: 2, under a stringent condition, and which encodesa protein having the carbamoyl-phosphate synthetase activity, from DNAcoding for carbamoyl-phosphate synthetase having mutation or from a cellharboring it. The “stringent condition” referred to herein is acondition under which so-called specific hybrid is formed, andnon-specific hybrid is not formed. It is difficult to clearly expressthis condition by using any numerical value. However, for example, thestringent condition includes a condition under which DNA's having highhomology, for example, DNA's having homology of not less than 70%,preferably not less than 80%, more preferably not less than 90% arehybridized with each other, and DNA's having homology lower than theabove are not hybridized with each other. Alternatively, the stringentcondition is exemplified by a condition under which DNA's are hybridizedwith each other at a salt concentration corresponding to an ordinarycondition of washing in Southern hybridization, i.e., 60° C., 1×SSC,0.1% SDS, preferably 0.1×SSC, 0.1% SDS.

[0060] As a probe, a partial sequence of the nucleotide sequence of SEQID NO: 1 can also be used. Such a probe may be prepared by PCR usingoligonucleotides produced based on the nucleotide sequence of SEQ ID NO:1 as primers, and a DNA fragment containing the nucleotide sequence ofSEQ ID NO: 1 as a template. When a DNA fragment in a length of about 300bp is used as the probe, the conditions of washing for the hybridizationconsist of, for example, 50° C., 2×SSC, and 0.1% SDS.

[0061] Because the nucleotide sequence of the DNA of the presentinvention has been elucidated, the DNA of the present invention can beobtained by amplifying it from coryneform bacterial chromosome DNAthrough polymerase chain reaction (PCR: polymerase chain reaction; seeWhite, T. J. et al., Trends Genet., 5, 185 (1989)) utilizingoligonucleotides prepared based on that nucleotide sequence as primers,or by selecting it from a coryneform bacterial chromosome DNA library byhybridization utilizing an oligonucleotide prepared based on thatnucleotide sequence as a probe. As nucleotide sequences of the primersused for PCR, a region upstream from the nucleotide number 283,preferably a region upstream from the nucleotide number 185 of SEQ IDNO: 1 can suitably be selected as the 5′ primer, and a region downstreamfrom the nucleotide number 4809 of SEQ ID NO: 1 can suitably be selectedas the 3′ primer.

[0062] Examples of the host for the expression of the DNA of the presentinvention include various bacteria such as Escherichia coli andcoryneform bacteria including Brevibacterium lactofermentum andBrevibacterium flavum, eukaryotic cells such as those of Saccharomycescerevisiae and so forth. In order to introduce the DNA of the presentinvention into these hosts, the host cells can be transformed with arecombinant vector obtained by inserting the DNA of the presentinvention into a vector selected according to the nature of the host inwhich the DNA is to be expressed. This procedure can be performed by amethod well known to those skilled in the art. Specific examples of themethod include the methods used for transformation of Escherichia colimentioned above, the method in which competent cells are prepared fromcells at the proliferating stage to introduce DNA, as reported forBacillus subtilis (Duncan, C. H., Wilson, G. A. and Young, F. E., Gene,1, 153 (1977)), the method in which DNA recipient cells are allowed tobe in a state of protoplasts or spheroplasts capable of incorporatingrecombinant DNA with ease to introduce recombinant DNA into the DNArecipient cells, as known for Bacillus subtilis, actinomycetes, andyeasts (Chang, S. and Choen, S. N., Molec. Gen. Genet., 168, 111 (1979);Bibb, M. J., Ward, J. M. and Hopwood, O. A., Nature, 274, 398 (1978);Hinnen, A., Hicks, J. B. and Fink, G. R., Proc. Natl. Acad. Sci. USA,75, 1929 (1978)), the electric pulse method useful for cryneformbacteria (refer to Japanese Patent Publication Laid-Open No. 2-207791)and so forth.

[0063] The DNA to be introduced into the host such as those mentionedabove may be DNA containing either carA or carB, or DNA containing bothof them. Further, in order to attain efficient expression of thesegenes, a promoter functioning in the host cells such as lac, trp andP_(L) may be ligated at a position upstream from carA or carB.

[0064] Carbamoyl-phosphate synthetase or its subunits can be produced byculturing a transformant such as those mentioned above under a conditionthat allows the expression of carA or carB. The DNA of the presentinvention can also be utilized for breeding of L-arginine-producingbacteria or nucleic acid-producing bacteria such as uracil-producingbacteria. That is, a transformant introduced with the DNA of the presentinvention, in particular, one introduced with either carA or carB orboth of them, should have increased carbamoyl-phosphate synthetaseactivity compared with non-transformants. Consequently, its productivityfor L-arginine or nucleic acid such as uracil is improved.

[0065] <2> Method for producing L-arginine According to the PresentInvention

[0066] L-Arginine can efficiently be produced by culturing amicroorganism that has enhanced intracellular carbamoyl-phosphatesynthetase activity, and has L-arginine productivity in a medium toproduce and accumulate L-arginine in the medium, and collecting theL-arginine from the medium.

[0067] Specific examples of the microorganism having L-arginineproductivity include coryneform bacteria, bacteria belonging to thegenera Bacillus, Serratia and Escherichia, yeast species belonging tothe genus Saccharomyces or Candida. Of these, coryneform bacteria arepreferred.

[0068] Exemplary specific species include Bacillus subtilis as abacterium belonging to the genus Bacillus, Serratia marcescens as abacterium belonging to the genus Serratia, Escherichia coli as abacterium belonging to the genus Escherichia, Saccharomyces cerevisiaeas a yeast species belonging to the genus Saccharomyces, Candidatropicalis as a yeast species belonging to the genus Candida and soforth.

[0069] Exemplary microorganisms having L-arginine productivity includeBacillus subtilis resistant to 5-azauracil, 6-azauracil, 2-thiouracil,5-fluorouracil, 5-bromouracil, 5-azacytosine and so forth, Bacillussubtilis resistant to arginine hydroxamate and 2-thiouracil, Bacillussubtilis resistant to arginine hydroxamate and 6-azauracil (see JapanesePatent Laid-open No. 49-1268191),

[0070]Bacillus subtilis resistant to histidine analogues or tryptophananalogues (see Japanese Patent Laid-open No. 52-114092),

[0071] a mutant of Bacillus subtilis exhibiting auxotrophy for at leastone of methionine, histidine, threonine, proline, isoleucine, lysine,adenine, guanine and uracil (or uracil precursor) (see Japanese PatentLaid-open No. 52-99289),

[0072]Bacillus subtilis resistant to arginine hydroxamate (see JapanesePatent Publication No. 51-6754),

[0073]Serratia marcescens exhibiting succinic acid auxotrophy orresistance to nucleic acid base analogues (Japanese Patent Laid-open No.58-9692),

[0074]Serratia marcescens deficient in ability to metabolize arginineand exhibiting resistance to arginine antagonists and canavanine andauxotorophy for lysine (see Japanese Patent Laid-open No. 52-8729),

[0075]Escherichia coli introduced with the argA gene (see JapanesePatent Laid-open No. 57-5693),

[0076]Saccharomyces cerevisiae resistant to arginine, argininehydroxamate, homoarginine, D-arginine and canavanine, or resistant toarginine hydroxamate and 6-azauracil (see Japanese Patent Laid-open No.53-143288),

[0077]Candida tropicalis resistant to canavanine (see Japanese PatentLaid-open No. 53-3586) and so forth.

[0078] Coryneform bacteria include those bacteria having been hithertoclassified into the genus Brevibacterium but united into the genusCorynebacterium at present (Int. J. Syst. Bacteriol., 41, 255 (1981)),and include bacteria belonging to the genus Brevibacterium closelyrelative to the genus Corynebacterium. Examples of such coryneformbacteria are listed below.

[0079]Corynebacterium acetoacidophilum

[0080]Corynebacterium acetoglutamicum

[0081]Corynebacterium alkanolyticum

[0082]Corynebacterium callunae

[0083]Corynebacterium glutamicum

[0084]Corynebacterium lilium (Corynebacterium glutamicum)

[0085]Corynebacterium melassecola

[0086]Corynebacterium thermoaminogenes

[0087]Corynebacterium herculis

[0088]Brevibacterium divaricatum

[0089] (Corynebacterium glutamicum)

[0090]Brevibacterium flavum (Corynebacterium glutamicum)

[0091]Brevibacterium immariophilum

[0092]Brevibacterium lactofermentum

[0093] (Corynebacterium glutamicum)

[0094]Brevibacterium roseum

[0095]Brevibacterium saccharolyticum

[0096]Brevibacterium thiogenitalis

[0097]Brevibacterium album

[0098]Brevibacterium cerinum

[0099]Microbacterium ammoniaphilum

[0100] The coryneform bacteria that have the L-arginine productivity arenot particularly limited so long as they have the L-arginineproductivity. They include, for example, wild-type strains of coryneformbacteria; coryneform bacteria resistant to certain agents includingsulfa drugs, 2-thiazolealanine, α-amino-β-hydroxyvaleric acid and thelike; coryneform bacteria exhibiting L-histidine, L-proline,L-threonine, L-isoleucine, L-methionine, or L-tryptophan auxotrophy inaddition to the resistance to 2-thiazolealanine (Japanese PatentLaid-open No. 54-44096); coryneform bacteria resistant to ketomalonicacid, fluoromalonic acid, or monofluoroacetic acid (Japanese PatentLaid-open No. 57-18989); coryneform bacteria resistant to argininol(Japanese Patent Laid-open No. 62-24075); coryneform bacteria resistantto X-guanidine (X represents a derivative of fatty acid or aliphaticchain, Japanese Patent Laid-open No. 2-186995) and so forth.

[0101] Specifically, the following bacterial strains can be exemplified.

[0102]Brevibacterium flavum AJ11169 (FERM BP-6892)

[0103]Brevibacterium lactofermentum AJ12092 (FERM BP-6906)

[0104]Brevibacterium flavum AJ11336 (FERM BP-6893)

[0105]Brevibacterium flavum AJ11345 (FERM BP-6893)

[0106]Brevibacterium lactofermentum AJ12430 (FERM BP-2228)

[0107] The AJ11169 strain and the AJ12092 strain are the2-thiazolealanine resistant strains mentioned in Japanese PatentLaid-open No. 54-44096, the AJ11336 strain is the strain havingargininol resistance and sulfadiazine resistance mentioned in JapanesePatent Publication No. 62-24075, the AJ11345 strain is the strain havingargininol resistance, 2-thiazolealanine resistance, sulfaguanidineresistance, and exhibiting histidine auxotrophy mentioned in JapanesePatent Publication No. 62-24075, and the AJ12430 strain is the strainhaving octylguanidine resistance and 2-thiazolealanine resistancementioned in Japanese Patent Laid-open No. 2-186995.

[0108] The intracellular carbamoyl-phosphate synthetase activity of suchmicroorganisms having the L-arginine productivity as mentioned above canbe enhanced by, for example, increasing copy number of a gene coding forthe carbamoyl-phosphate synthetase in the cells of the aforementionedmicroorganisms. The enhancement of the carbamoyl-phosphate synthetaseactivity can also be achieved by, in addition to the aforementioned geneamplification, modifying an expression regulation sequence for the DNAcoding for carbamoyl-phosphate synthetase so that expression of the DNAgene coding for carbamoyl-phosphate synthetase should be enhanced.Specifically, an expression regulation sequence such as a promoter for agene coding for carbamoyl-phosphate synthetase on the chromosomal DNA ora plasmid can be replaced with a stronger one (see Japanese PatentLaid-open No. 1-215280). Strong promoters, which function in cells ofcoryneform bacteria, include lac promoter, tac promoter, trp promoter,of Escherichia coli (Y. Morinaga, M. Tsuchiya, K. Miwa and K. Sano, J.Biotech., 5, 305-312 (1987)) and the like. In addition, trp promoter ofCorynebacterium bacteria is also a preferable promoter (Japanese PatentLaid-open No. 62-195294). By the replacement with these promoters thecarbamoyl-phosphate synthetase activity is enhanced. The modification ofexpression regulation sequence may be combined with the increasing ofthe copy number of DNA coding for carbamoyl-phosphate synthetase.Further, the intracellular carbamoyl-phosphate synthetase activity canbe enhanced by introducing one or more mutations into the enzyme proteinof carbamoyl-phosphate synthetase so that the specific activity of theenzyme should be increased.

[0109] Examples of the DNA coding for carbamoyl-phosphate synthetaseinclude the aforementioned carA and carb genes of Brevibacteriumlactofermentum and one containing both of them.

[0110] Examples of the vector for introducing DNA coding forcarbamoyl-phosphate synthetase into a microorganism include vectorsautonomously replicable in cells of the microorganism. Specifically, theaforementioned vectors autonomously replicable in Escherichia colicells, and the vectors autonomously replicable in both of Escherichiacoli cells and coryneform bacterium cells.

[0111] The medium used for culturing a microorganism having enhancedintracellular carbamoyl-phosphate synthetase activity and L-arginineproductivity obtained as described above may be a well-known mediumconventionally used for the production of amino acids by fermentation.That is, it is a usual medium that contains a carbon source, nitrogensource, inorganic ions, and other organic components as required.

[0112] As the carbon source, it is possible to use sugars such asglucose, sucrose, lactose, galactose, fructose and starch hydrolysates;alcohols such as glycerol and sorbitol; or organic acids such as fumaricacid, citric acid and succinic acid and so forth.

[0113] As the nitrogen source, it is possible to use inorganic ammoniumsalts such as ammonium sulfate, ammonium chloride and ammoniumphosphate, organic nitrogen such as soybean hydrolysates, ammonia gas,aqueous ammonia and so forth.

[0114] The medium preferably contains a suitable amount of requiredsubstance such as vitamin B₁ and L-homoserine, yeast extract and soforth as trace amount organic nutrients. Other than those substances, asmall amount of potassium phosphate, magnesium sulfate, iron ions,manganese ions and so forth may be added to the medium.

[0115] The cultivation is preferably performed under an aerobiccondition for 1-7 days. Cultivation temperature is preferably 24-37° C.,and pH of the medium during the cultivation is preferably 5-9. Inorganicor organic acidic or alkaline substances, ammonia gas and so forth maybe used for adjusting pH. L-Arginine can usually be recovered from thefermentation medium by a combination of known techniques such as ionexchange resin method.

BEST MODE FOR CARRYING OUT THE INVENTION

[0116] Hereafter, the present invention will be explained morespecifically with reference to the following examples.

EXAMPLE 1 Cloning of carA and carB of Brevibacterium lactofermentum

[0117] <1> Preparation of Chromosome DNA of Brevibacteriumlactofermentum ATCC13869

[0118]Brevibacterium lactofermentum ATCC13869 was inoculated to 100 mlof T-Y culture medium (1% of Bacto-Trypton (Difco), 0.5% of Bacto-YeastExtract (Difco), 0.5% of NaCl (pH 7.2)), and cultured at a temperatureof 31.5° C. for 8 hours to obtain a culture. The culture was centrifugedat 3,000 r.p.m. for 15 minutes to obtain 0.5 g of wet bacterial cells,and chromosome DNA was obtained from the bacterial cells according tothe method of Saito and Miura (Biochem. Biophys. Acta., 72, 619 (1963)).Then, 60 μg of the chromosome DNA and 3 units of restriction enzymeSau3AI were each mixed in 10 mM Tris-HCl buffer (containing 50 mM NaCl,10 mM MgSO₄ and 1 mM dithiothreitol (pH 7.4)), and allowed to react at atemperature of 37° C. for 30 minutes. The reaction mixture was subjectedto phenol extraction and ethanol precipitation in a conventional mannerto obtain 50 μg of chromosome DNA fragments of Brevibacteriumlactofermentum ATCC13869 digested with Sau3AI.

[0119] <2> Preparation of Gene Library of Brevibacterium lactofermentumATCC13869 using Plasmid Vector DNA

[0120] As a plasmid vector DNA autonomously replicable in both ofEscherichia coli cells and coryneform bacterium cells, pSAC4 was used.pSAC4 was prepared as follows. In order to make a vector pHSG399 forEscherichia coli (Takara Shuzo) autonomously replicable in coryneformbacterium cells, a replication origin of the previously obtained plasmidpHM1519 autonomously replicable in coryneform bacterium cells (Miwa, K.et al., Agric. Biol. Chem., 48 (1984) 2901-2903) was introduced into thevector (Japanese Patent Laid-open No. 5-7491). Specifically, pHM1519 wasdigested with restriction enzymes BamHI and KpnI to obtain a genefragment containing the replication origin, and the obtained fragmentwas blunt-ended by using Blunting Lit produced by Takara Shuzo, andinserted into the SalI site of pHSG399 using a SalI linker (produced byTakara Shuzo) to obtain pSAC4.

[0121] In 50 mM Tris-HCl buffer (containing 100 mM NaCl and 10 mMmagnesium sulfate (pH 7.4)), 20 μg of pSAC4 and 200 units of arestriction enzyme BamHI were mixed, and allowed to react at atemperature of 37° C. for 2 hours to obtain a digestion solution. Thissolution was subjected to phenol extraction and ethanol precipitation ina conventional manner. Then, in order to inhibit religation of the DNAfragments derived from the plasmid vector, the DNA fragments weredephosphorylated with bacterial alkaline phosphatase according to themethod described in Molecular Cloning, 2nd Edition (J. Sambrook, E. F.Fritsch and T. Maniatis, Cold Spring Harbor Laboratory Press, p1.56(1989)), and subjected to phenol extraction and ethanol precipitation ina conventional manner.

[0122] To 66 mM Tris-HCl buffer (pH 7.5) containing 66 mM magnesiumchloride, 10 mM dithiothreitol and 10 mM ATP, 1 μg of the pSAC4 digestedwith BamHI, 1 μg of the chromosome DNA fragments of Brevibacteriumlactofermentum ATCC13869 digested with Sau3AI obtained in Example 1, and2 units of T4 DNA ligase (produced by Takara Shuzo) were added, andallowed to react at a temperature of 16° C. for 16 hours to ligate theDNA. Then, Escherichia coli DH5 was transformed with this DNA mixture ina conventional manner, and plated on an L agar medium containing 170μg/ml of chloramphenicol to obtain about 20,000 colonies, which wereused as a gene library.

[0123] <3> Transformation of carB-deficient Strain of Escherichia coli(JEF8)

[0124] The carb-deficient strain of Escherichia coli, JEF8 (thr⁻31,ΔcarB, relA⁻, metB1; Mol. Gen. Genet., 133, 299 (1974)) was transformedwith a recombinant DNA mixture of the aforementioned gene library in aconventional manner. Transformants of about 15000 strains were obtainedas Cm resistant strains. These transformants were replicated on aminimum medium (5 g/L of glucose, 12.8 g/L of Na₂HPO₄, 3 g/L of KH₂PO₄,0.5 g/L of NaCl, 1 g/L of NH₄Cl, 40 μg/ml of L-threonine, 40 μg/ml ofL-methionine) not containing arginine and uracil, and the minimum mediumnot containing L-arginine, but containing only 50 μg/ml of uracil, andscreened for a strain in which arginine auxotrophy and uracil auxotrophywere restored, or a strain in which arginine auxotrophy was restored.Strains in which arginine auxotrophy was restored recovered both ofarginine auxotrophy and uracil auxotrophy. A plasmid harbored in one ofsuch strains was designated as p19, and the strain harboring it wasdesignated as JEF8/p19. The structure of p19 is shown in FIG. 1.

[0125] The Escherichia coli JEF8/p19 was designated as Escherichia coliAJ13574, and it was deposited at the National Institute of Bioscienceand Human-Technology, Agency of Industrial Science and Technology,Ministry of International Trade and Industry (postal code 305-8566, 1-3Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) on Jan. 28, 1999, andreceived an accession number of FERM P-17180, and transferred from theoriginal deposit to international deposit based on Budapest Treaty onJan. 6, 20000, and has been deposited as deposition number of FERMBP-6989.

[0126] <4> Acquisition of Plasmid Complementing Arginine and UracilAuxotrophy

[0127] A plasmid was prepared from JEF8/p19 in a conventional manner,and used for re-transformation of the JEF8 strain. The obtainedtransformants could grow in the minimum culture medium not containingL-arginine and uracil, and its auxotrophy for both of L-arginine anduracil was restored. Therefore, it was found that that the plasmidcontained a gene complementing the auxotrophy for both of L-arginine anduracil caused by deletion of carB in the Escherichia coli strain.

[0128] Further, this plasmid was introduced into the carA mutant ofEscherichia coli, RC50 (carA50, tsx⁻273, λ⁻, rpsL135 (str^(R)), malT1(λR), xylA7, thi⁻¹ ; Mol. Gen. Genet., 133, 299 (1974)). Since thestrain introduced with the plasmid was able to grow in the minimumculture medium not containing arginine and uracil, the plasmid was alsofound to have a gene complementing the auxotrophy for both of L-arginineand uracil caused by carA mutation of the Escherichia coli strain.

[0129] <5> Nucleotide Sequence Analysis of p19

[0130] Among the DNA sequence of p19, the nucleotide sequence of about4.8 kb from the HindIII side of the multi-cloning site of the vector tothe HindIII site contained in the insertion DNA fragment was determined.The nucleotide sequencing was performed by using Rohdamin TerminatorCycle Sequencing Kit (produced by ABI) according to the method ofSanger. The obtained nucleotide sequence is shown as SEQ ID NO: 1 inSequence Listing. From analysis of a consensus sequence which located inthe upstream region of this gene, it was estimated that two open readingframes (open reading frame from 283rd G to 1461st A and open readingframe from 1756th G to 4809th T) were contained in this sequence. Thenucleotides of the 162nd (TGCATA) to 194th (TATAAT), the 185th (TGCATA)to 213rd (TAAACT), the 203rd (TTGAAT) 230th (TATCAA), or the 224th(TTATCA) to 251st (TAAAAA) can be estimated to be a promoter region forregulating the transcription.

[0131] The amino acid sequences encoded by these open reading frames arerepresented with the nucleotide sequences. The amino acid sequences werealso shown in SEQ ID NOS: 2 and 3. A protein database (GenBank CDS) wassearched for sequences exhibiting homology with these amino acidsequences. As a result, it was found that the 5′ open reading frameshowed high homology (about 40%) with carA gene products of Escherichiacoli, Bacillus subtilis and so forth, and the 3′ open reading frameshowed high homology with known carB gene products of Escherichia coli,Bacillus stearothermophilus and so forth (about 40 to 50%). Therefore,it was suggested that these open reading frames coded for carA and carB,respectively.

[0132] <6> Introduction of carA and carB into Wild-Type Strain ofCoryneform Bacteria

[0133] p19 was introduced into the Brevibacterium flavum wild strain2247 (AJ14067) by the electric pulse method (Japanese Patent Laid-openNo. 2-207791). The transformants were selected as chloramphenicolresistant strains on a CM2G plate medium (containing 10 g ofpolypeptone, 10 g of yeast extract, 5 g of glucose, 5 g of NaCl, 15 g ofagar in 1 L of pure water, pH 7.2) containing 5 μg/ml of chloramphenicolto obtain 2247/p19.

[0134] EXAMPLE 2

Production of L-arginine by Coryneform Bacteria Introduced with carA andcarB

[0135] <1> Preparation of Shuttle Vector

[0136] First, a plasmid vector autonomously replicable in both ofEscherichia coli cells and coryneform bacterium cells was newly producedas a plasmid used for introducing the carA and carB genes intocoryneform bacteria.

[0137] A vector containing a drug resistance gene of Streptococcusfaecalis was constructed first. The kanamycin resistant gene ofStreptococcus faecalis was amplified by PCR from a known plasmidcontaining that gene. The nucleotide sequence of the kanamycin resistantgene of Streptococcus faecalis has already been clarified (Trieu-Cuot,P. and Courvalin, P., Gene, 23(3), 331-341 (1983)). The primers shown asSEQ ID NOS: 4 and 5 were synthesized based on that sequence, and PCR wasperformed by using pDG783 (Anne-Marie Guerout-Fleury et al., Gene, 167,335-337 (1995)) as a template to amplify a DNA fragment containing thekanamycin resistant gene and its promoter.

[0138] The obtained DNA fragment was purified by SUPREC02 produced bythe Takara Shuzo, then fully digested with restriction enzymes HindIIIand HincII, and blunt-ended. The blunt-ending was attained by usingBlunting Kit produced by Takara Shuzo. This DNA fragment was mixed withand ligated to a DNA fragment, which had been obtained by performing PCRusing the primers shown as SEQ ID NOS: 6 and 7 and pHSG399 (see S.Takeshita et al., Gene, 61, 63-74 (1987)) as a template, purifying andblunt-ending the resulted amplification product. The ligation reactionwas performed by DNA Ligation Kit ver. 2 produced by Takara Shuzo.Competent cells of Escherichia coli JM109 (produced by Takara Shuzo)were transformed with the ligated DNA, plated on L madium (10 g/L ofBacto-trypton, 5 g/L of Bacto-yeast extract, 5 g/L of NaCl, 15 g/L ofagar, pH 7.2) containing 10 μg/ml of IPTG(isopropyl-β-D-thiogalactopyranoside), 40 μg/ml of X-Gal(5-bromo-4-chloro-3-indolyl-β-D-galactoside) and 25 μg/ml of kanamycin,and cultured overnight. The emerged blue colonies were picked up, andseparated into single colonies to obtain transformant strains.

[0139] Plasmids were prepared from the transformant strains by thealkali method (Text for Bioengineering Experiments, Edited by theSociety for Bioscience and Bioengineering, Japan, p.105, Baifukan,1992), and restriction maps were prepared. One having a restriction mapequivalent to that of FIG. 2 was designated as pK1. This plasmid isstably retained in Escherichia coli, and imparts kanamycin resistance toa host. Moreover, since it contains the lacZ′ gene, it is suitably usedas a cloning vector.

[0140] The plasmid pAM330 extracted from Brevibacterium lactofermentumATCC13869 (see Japanese Patent Laid-open No. 58-67699) was fullydigested with a restriction enzyme HindIII, and blunt-ended. Thisfragment was ligated to a fragment obtained by fully digesting theaforementioned pK1 with a restriction enzyme BsaAI. Brevibacteriumlactofermentum ATCC13869 was transformed with the ligated DNA. Thetransformation was performed by the electric pulse method (see JapanesePatent Laid-open No. 2-207791). Transformants were selected on a M-CM2Bplate (10 g/L of polypeptone, 10 g/L of yeast extract, 5 g/L of NaCl, 10μg/L of biotin, 15 g/L of agar, pH 7.2) containing 25 μg/ml ofkanamycin. After cultivation for 2 days, colonies were picked up, andseparated into single colonies to obtain the transformants. Plasmid DNAwas prepared from the transformants, and restriction maps were prepared.One having the same restriction map as that of FIG. 3 was designated aspSFK6. This plasmid can autonomously replicate in both of Escherichiacoli and coryneform bacteria, and imparts kanamycin resistance to ahost.

[0141] <2> Introduction of carA and carB genes into Coryneform Bacteriaand Production of L-arginine

[0142] The aforementioned pSFK6 was digested with SmaI and HindIII. Theproduct was ligated to carA and carB gene fragments, which had beenobtained by digesting the plasmid p19 prepared from JEF8/p19F in aconventional manner with a restriction enzyme XbaI, blunt-ending theproduct by using Blunting Kit produced by Takara Shuzo, and furtherdigesting the product with a restriction enzyme HindIII, to obtain aplasmid pcarAB, which contained the carA and carB genes and couldautonomously replicate in coryneform bacteria.

[0143] pcarAB was introduced into Brevibacterium flavum AJ11345 andAJ11336 by the electric pulse method (Japanese Patent Laid-open No.2-207791). Transformants were selected on a M-CM2B plate (10 g/L ofpolypeptone, 10 g/L of yeast extract, 5 g/L of glucose, 5 g/L of NaCl,15 g/L of agar, pH 7.2) containing 25 μg/ml of kanamycin as kanamycinresistant strains. As control, transformants were obtained by similarlyintroducing pSFK6 into AJ11345 and AJ11336.

[0144] Each of the aforementioned transformants was plated on an agarmedium containing 0.5 g/dl of glucose, 1 g/dl of polypeptone, 1 g ofyeast extract, 0.5 g/dl of NaCl and 5 μg/l of chloramphenicol, andcultured at 31.5° C. for 20 hours. One inoculating loop of the obtainedcells were inoculated to a medium containing 4 g/dl of glucose, 6.5 g/dLof ammonium sulfate, 0.1 g/dl of KH₂PO₄, 0.04 g/dl of MgSO₄, 0.001 g/dlof FeSO₄, 0.01 g/dl of MnSO₄, 5 μg/dl of VB₁, 5 μg/dl of biotin, 45mg/dl of soybean hydrolysates (as an amount of N), and cultured in aflask at 31.5° C. for 50 hours with shaking. The amounts of L-arginineproduced by each strain were shown in Table 1.

[0145] The strains introduced with the carA and carB gene showedimproved L-arginine productivity compared with the strains introducedonly with the vector. TABLE 1 Strain/plasmid L-arginine (g/dl)AJ11345/pSFK6 1.33 AJ11345/pcarAB 1.39 AJ11336/pSFK6 0.71 AJ11336/pcarAB0.79

[0146]

1 7 1 4837 DNA Brevibacterium lactofermentum CDS (283)..(1461) 1gatccaggaa aaacctggac agcatccggt gcagactttg cgtccaaggc tgaaaacacc 60ccatttgagg gccaggaatt cagcgctaag gtcacacaca ccgtgcttcg tggcaaggtg 120acttgtgcag acggagttgc gcaagacgct taacgggtgg gtgcatagta tgcacgcgcc 180gcattgcata taatgcaatg aattgaataa actacattca gggttatcaa ccagccaatt 240tcttttaaaa agacagacac acgaaaggcg acaacagtca cc gtg agt aaa gac 294 ValSer Lys Asp 1 acc acc acc tac cag gga gtc acc gag atc gga tcc gtt ccggca tac 342 Thr Thr Thr Tyr Gln Gly Val Thr Glu Ile Gly Ser Val Pro AlaTyr 5 10 15 20 ctg gtt ctt gca gac gga cgt acc ttc acc gga ttt ggc tttgga gct 390 Leu Val Leu Ala Asp Gly Arg Thr Phe Thr Gly Phe Gly Phe GlyAla 25 30 35 atc ggc acc acc ctt ggt gag gca gtg ttc acc acc gcc atg accggt 438 Ile Gly Thr Thr Leu Gly Glu Ala Val Phe Thr Thr Ala Met Thr Gly40 45 50 tac caa gaa acc atg acc gat cct tcc tat cac cgc cag att gtt gtg486 Tyr Gln Glu Thr Met Thr Asp Pro Ser Tyr His Arg Gln Ile Val Val 5560 65 gct acc gca cca cag atc ggt aac acc ggc tgg aac gat gag gac aac534 Ala Thr Ala Pro Gln Ile Gly Asn Thr Gly Trp Asn Asp Glu Asp Asn 7075 80 gag tcc cgc gac ggc aag att tgg gtt gca ggc ctt gtt atc cgc gac582 Glu Ser Arg Asp Gly Lys Ile Trp Val Ala Gly Leu Val Ile Arg Asp 8590 95 100 ctc gca gca cgt gtg tcc aac tgg cgc gcc acc acc tcc ttg cagcag 630 Leu Ala Ala Arg Val Ser Asn Trp Arg Ala Thr Thr Ser Leu Gln Gln105 110 115 gaa atg gca gac caa ggc atc gtc ggc atc ggc gga atc gac acccgc 678 Glu Met Ala Asp Gln Gly Ile Val Gly Ile Gly Gly Ile Asp Thr Arg120 125 130 gca ctg gtt cgc cac ctg cgc aac gaa ggt tcc atc gca gcg ggcatc 726 Ala Leu Val Arg His Leu Arg Asn Glu Gly Ser Ile Ala Ala Gly Ile135 140 145 ttc tcc ggc gct gac gca cag cgc cca gtt gaa gaa ctc gta gagatc 774 Phe Ser Gly Ala Asp Ala Gln Arg Pro Val Glu Glu Leu Val Glu Ile150 155 160 gtc aag aat cag cca gca atg acc ggc gca aac ctc tcc gtt gaggtc 822 Val Lys Asn Gln Pro Ala Met Thr Gly Ala Asn Leu Ser Val Glu Val165 170 175 180 tct gct gat gaa acc tac gtc atc gaa gct gag ggc gaa gagcgc cac 870 Ser Ala Asp Glu Thr Tyr Val Ile Glu Ala Glu Gly Glu Glu ArgHis 185 190 195 acc gtc gtg gcc tac gac ctg ggc att aag caa aac acc ccacgt cgt 918 Thr Val Val Ala Tyr Asp Leu Gly Ile Lys Gln Asn Thr Pro ArgArg 200 205 210 ttc tct gca cgc ggt gtt cgc acc gtc atc gtg cct gct gaaacc cca 966 Phe Ser Ala Arg Gly Val Arg Thr Val Ile Val Pro Ala Glu ThrPro 215 220 225 ttg gag gac atc aag cag tac aac cca tca ggc gtg ttt atctcc aat 1014 Leu Glu Asp Ile Lys Gln Tyr Asn Pro Ser Gly Val Phe Ile SerAsn 230 235 240 ggc cct ggc gac cct gca gca gca gac gtc atg gtt gat atcgtc cgc 1062 Gly Pro Gly Asp Pro Ala Ala Ala Asp Val Met Val Asp Ile ValArg 245 250 255 260 gaa gtt ctg gaa gcc gac att cca ttc ttt ggc atc tgcttc ggc aac 1110 Glu Val Leu Glu Ala Asp Ile Pro Phe Phe Gly Ile Cys PheGly Asn 265 270 275 cag atc ctc ggc cgc gca ttc ggc atg gag acc tac aagctg aag ttc 1158 Gln Ile Leu Gly Arg Ala Phe Gly Met Glu Thr Tyr Lys LeuLys Phe 280 285 290 ggc cac cgc ggc atc aac gtt cca gtg aag aac cac atcacc ggc aag 1206 Gly His Arg Gly Ile Asn Val Pro Val Lys Asn His Ile ThrGly Lys 295 300 305 atc gac atc acc gcc cag aac cac ggc ttc gca ctc aagggt gaa gca 1254 Ile Asp Ile Thr Ala Gln Asn His Gly Phe Ala Leu Lys GlyGlu Ala 310 315 320 ggc cag gaa ttc gag aca gat ttc ggc act gcg att gtcacc cac acc 1302 Gly Gln Glu Phe Glu Thr Asp Phe Gly Thr Ala Ile Val ThrHis Thr 325 330 335 340 tgc ctt aac gac ggc gtc gtt gaa ggt gtt gcg ctgaag tcc gga cgc 1350 Cys Leu Asn Asp Gly Val Val Glu Gly Val Ala Leu LysSer Gly Arg 345 350 355 gca tac tcc gtt cag tac cac cca gag gcc gct gccggc cca aat gat 1398 Ala Tyr Ser Val Gln Tyr His Pro Glu Ala Ala Ala GlyPro Asn Asp 360 365 370 gca agc ccc ctg ttt gac cag ttt gtt gag ctg atggat gca gac gct 1446 Ala Ser Pro Leu Phe Asp Gln Phe Val Glu Leu Met AspAla Asp Ala 375 380 385 cag aag aaa ggc gca taaataac atg cca aag cgt tcagat att aac cac 1496 Gln Lys Lys Gly Ala Met Pro Lys Arg Ser Asp Ile AsnHis 390 395 400 gtc ctc gtc atc ggt tcc ggc ccc atc gtc att ggc cag gcatgt gaa 1544 Val Leu Val Ile Gly Ser Gly Pro Ile Val Ile Gly Gln Ala CysGlu 405 410 415 ttc gac tac tcc ggc acc cag gct tgc cgc gtg ctg aag gaagag gga 1592 Phe Asp Tyr Ser Gly Thr Gln Ala Cys Arg Val Leu Lys Glu GluGly 420 425 430 ctg cgc gtc acc ctc atc aac tcc aac cca gca acg atc atgacc gac 1640 Leu Arg Val Thr Leu Ile Asn Ser Asn Pro Ala Thr Ile Met ThrAsp 435 440 445 450 cca gaa atg gct gac cac acc tac gtg gag cca atc gagccg gaa tac 1688 Pro Glu Met Ala Asp His Thr Tyr Val Glu Pro Ile Glu ProGlu Tyr 455 460 465 atc gac aag att ttc gct aag gag atc gag cag ggc caccca atc gac 1736 Ile Asp Lys Ile Phe Ala Lys Glu Ile Glu Gln Gly His ProIle Asp 470 475 480 gcc gtc ctg gca acc ctt ggt ggc cag act gca ctt aacgca gct atc 1784 Ala Val Leu Ala Thr Leu Gly Gly Gln Thr Ala Leu Asn AlaAla Ile 485 490 495 cag ctg gat cgc ctc ggc atc ctg gaa aag tac ggc gttgaa ctc atc 1832 Gln Leu Asp Arg Leu Gly Ile Leu Glu Lys Tyr Gly Val GluLeu Ile 500 505 510 ggt gca gac atc gat gcc att gag cgc ggc gaa gat cgccag aag ttc 1880 Gly Ala Asp Ile Asp Ala Ile Glu Arg Gly Glu Asp Arg GlnLys Phe 515 520 525 530 aag gat att gtc acc acc atc ggt ggc gaa tcc gcgcgt tcc cgc gtc 1928 Lys Asp Ile Val Thr Thr Ile Gly Gly Glu Ser Ala ArgSer Arg Val 535 540 545 tgc cac aac atg gac gaa gtc cat gag act gtc gcagaa ctt ggc ctt 1976 Cys His Asn Met Asp Glu Val His Glu Thr Val Ala GluLeu Gly Leu 550 555 560 cca gta gtc gtg cgt cca tcc ttc act atg ggt ggcctg ggc tcc ggt 2024 Pro Val Val Val Arg Pro Ser Phe Thr Met Gly Gly LeuGly Ser Gly 565 570 575 ctt gca tac aac acc gaa gac ctt gag cgc atc gcaggt ggc gga ctt 2072 Leu Ala Tyr Asn Thr Glu Asp Leu Glu Arg Ile Ala GlyGly Gly Leu 580 585 590 gct gca tct cct gaa gca aac gtc ttg atc gaa gaatcc atc ctt ggt 2120 Ala Ala Ser Pro Glu Ala Asn Val Leu Ile Glu Glu SerIle Leu Gly 595 600 605 610 tgg aag gaa ttc gag ctc gag ctc atg cgc gatacc gca gac aac gtt 2168 Trp Lys Glu Phe Glu Leu Glu Leu Met Arg Asp ThrAla Asp Asn Val 615 620 625 gtg gtt atc tgc tcc att gaa aac gtc gac gcactg ggc gtg cac acc 2216 Val Val Ile Cys Ser Ile Glu Asn Val Asp Ala LeuGly Val His Thr 630 635 640 ggc gac tct gtc acc gtg gca cct gcc ctg accctg act gac cgt gaa 2264 Gly Asp Ser Val Thr Val Ala Pro Ala Leu Thr LeuThr Asp Arg Glu 645 650 655 ttc cag aag atg cgc gat cag ggt atc gcc atcatc cgc gag gtc ggc 2312 Phe Gln Lys Met Arg Asp Gln Gly Ile Ala Ile IleArg Glu Val Gly 660 665 670 gtg gac acc ggt gga tgt aac atc cag ttc gctatc aac cca gtt gat 2360 Val Asp Thr Gly Gly Cys Asn Ile Gln Phe Ala IleAsn Pro Val Asp 675 680 685 690 ggc cgc atc atc acc att gag atg aac ccacgt gtg tct cgt tcc tcc 2408 Gly Arg Ile Ile Thr Ile Glu Met Asn Pro ArgVal Ser Arg Ser Ser 695 700 705 gcg ctg gca tcc aag gca acg ggc ttc ccaatt gcc aag atg gct gcc 2456 Ala Leu Ala Ser Lys Ala Thr Gly Phe Pro IleAla Lys Met Ala Ala 710 715 720 aag ctg gct atc gga tac acc ctg gat gagatc acc aac gac atc act 2504 Lys Leu Ala Ile Gly Tyr Thr Leu Asp Glu IleThr Asn Asp Ile Thr 725 730 735 ggt gaa acc cca gct gcg ttt gag ccc accatc gac tac gtc gtg gtc 2552 Gly Glu Thr Pro Ala Ala Phe Glu Pro Thr IleAsp Tyr Val Val Val 740 745 750 aag gcc cca cgc ttt gct ttc gag aag tttgtc ggc gct gat gac act 2600 Lys Ala Pro Arg Phe Ala Phe Glu Lys Phe ValGly Ala Asp Asp Thr 755 760 765 770 ttg acc acc acc atg aag tcc gtc ggtgag gtc atg tcc ctg ggc cgt 2648 Leu Thr Thr Thr Met Lys Ser Val Gly GluVal Met Ser Leu Gly Arg 775 780 785 aac tac att gca gca ctg aac aag gcactg cgt tcc ctg gaa acc aag 2696 Asn Tyr Ile Ala Ala Leu Asn Lys Ala LeuArg Ser Leu Glu Thr Lys 790 795 800 cag cag ggt ttc tgg acc aag cct gatgag ttc ttc gca ggg gag cgc 2744 Gln Gln Gly Phe Trp Thr Lys Pro Asp GluPhe Phe Ala Gly Glu Arg 805 810 815 gct acc gat aag gca gct gtt ctg gaagat ctc aag cgc cca acc gaa 2792 Ala Thr Asp Lys Ala Ala Val Leu Glu AspLeu Lys Arg Pro Thr Glu 820 825 830 ggc cgc ctc tac gac gtt gag ctg gcaatg cgc ctt ggc gca agc gtg 2840 Gly Arg Leu Tyr Asp Val Glu Leu Ala MetArg Leu Gly Ala Ser Val 835 840 845 850 gaa gaa ctc tac gaa gca tct tctatt gat cct tgg ttc ctc gcc gag 2888 Glu Glu Leu Tyr Glu Ala Ser Ser IleAsp Pro Trp Phe Leu Ala Glu 855 860 865 ctt gaa gct ctc gtg cag ttc cgccag aag ctc gtt gac gca cca ttc 2936 Leu Glu Ala Leu Val Gln Phe Arg GlnLys Leu Val Asp Ala Pro Phe 870 875 880 ctc aac gaa gat ctc ctg cgc gaagca aag ttc atg ggt ctg tcc gac 2984 Leu Asn Glu Asp Leu Leu Arg Glu AlaLys Phe Met Gly Leu Ser Asp 885 890 895 ctg cag atc gca gcc ctt cgc ccagag ttc gct ggc gaa gac ggc gta 3032 Leu Gln Ile Ala Ala Leu Arg Pro GluPhe Ala Gly Glu Asp Gly Val 900 905 910 cgc acc ttg cgt ctg tcc cta ggcatc cgc cca gta ttc aag act gtg 3080 Arg Thr Leu Arg Leu Ser Leu Gly IleArg Pro Val Phe Lys Thr Val 915 920 925 930 gat acc tgt gca gca gag tttgaa gct aag act ccg tac cac tac tcc 3128 Asp Thr Cys Ala Ala Glu Phe GluAla Lys Thr Pro Tyr His Tyr Ser 935 940 945 gca tac gag ctg gat cca gcagct gag tct gag gtc gca cca cag act 3176 Ala Tyr Glu Leu Asp Pro Ala AlaGlu Ser Glu Val Ala Pro Gln Thr 950 955 960 gag cgt gaa aag gtc ctg atcttg ggc tcc ggt cca aac cgc atc ggc 3224 Glu Arg Glu Lys Val Leu Ile LeuGly Ser Gly Pro Asn Arg Ile Gly 965 970 975 cag ggc atc gag ttc gac tattcc tgt gtt cac gca gct ctt gag ctc 3272 Gln Gly Ile Glu Phe Asp Tyr SerCys Val His Ala Ala Leu Glu Leu 980 985 990 tcc cgc gtc ggc tac gaa actgtc atg gtc aac tgc aac cca gag 3317 Ser Arg Val Gly Tyr Glu Thr Val MetVal Asn Cys Asn Pro Glu 995 1000 1005 acc gtg tcc acc gac tac gac accgct gac cgc ctg tac ttc gag 3362 Thr Val Ser Thr Asp Tyr Asp Thr Ala AspArg Leu Tyr Phe Glu 1010 1015 1020 cca ctg acc ttc gaa gac gtc atg gaggtc tac cac gct gag gcg 3407 Pro Leu Thr Phe Glu Asp Val Met Glu Val TyrHis Ala Glu Ala 1025 1030 1035 cag tcc ggc acc gtc gca ggt gtt atc gtccag ctt ggt ggc cag 3452 Gln Ser Gly Thr Val Ala Gly Val Ile Val Gln LeuGly Gly Gln 1040 1045 1050 act cct ctg ggc ttg gca gat cgt ttg aag aaggct ggc gtc cct 3497 Thr Pro Leu Gly Leu Ala Asp Arg Leu Lys Lys Ala GlyVal Pro 1055 1060 1065 gtc att ggt acc tcc cca gag gca atc gac atg gctgag gac cgt 3542 Val Ile Gly Thr Ser Pro Glu Ala Ile Asp Met Ala Glu AspArg 1070 1075 1080 ggc gag ttc ggt gca ctg ctg aac cgc gag cag ctt cctgct cca 3587 Gly Glu Phe Gly Ala Leu Leu Asn Arg Glu Gln Leu Pro Ala Pro1085 1090 1095 gca ttc ggc acc gca acc tct ttc gaa gag gct cgc aca gtagcc 3632 Ala Phe Gly Thr Ala Thr Ser Phe Glu Glu Ala Arg Thr Val Ala1100 1105 1110 gat gag atc agc tac cca gtg ctg gtt cgc cct tcc tac gtcttg 3677 Asp Glu Ile Ser Tyr Pro Val Leu Val Arg Pro Ser Tyr Val Leu1115 1120 1125 ggt ggc cgt ggc atg gag att gtc tac gat gag gct tcc ctcgag 3722 Gly Gly Arg Gly Met Glu Ile Val Tyr Asp Glu Ala Ser Leu Glu1130 1135 1140 gat tac atc aac cgc gca act gag ttg tct tct gac cac ccagtg 3767 Asp Tyr Ile Asn Arg Ala Thr Glu Leu Ser Ser Asp His Pro Val1145 1150 1155 ctg gtt gac cgc ttc ctg gac aac gct att gag atc gac gtcgac 3812 Leu Val Asp Arg Phe Leu Asp Asn Ala Ile Glu Ile Asp Val Asp1160 1165 1170 gca ctg tgc gac ggc gac gaa gtc tac ctg gcg ggc gtc atggaa 3857 Ala Leu Cys Asp Gly Asp Glu Val Tyr Leu Ala Gly Val Met Glu1175 1180 1185 cac atc gag gaa gcc ggc att cac tcc ggt gac tcc gca tgtgca 3902 His Ile Glu Glu Ala Gly Ile His Ser Gly Asp Ser Ala Cys Ala1190 1195 1200 ctt cct cca atg act ttg ggc gca cag gac atc gag aag gtccgc 3947 Leu Pro Pro Met Thr Leu Gly Ala Gln Asp Ile Glu Lys Val Arg1205 1210 1215 gaa gca acc aag aag ctg gct ctg ggc atc ggc gta cag ggcctg 3992 Glu Ala Thr Lys Lys Leu Ala Leu Gly Ile Gly Val Gln Gly Leu1220 1225 1230 atg aac gtc cag tac gca ctc aag gac gac atc ctc tac gtcatc 4037 Met Asn Val Gln Tyr Ala Leu Lys Asp Asp Ile Leu Tyr Val Ile1235 1240 1245 gag gca aac cca cgt gca tcc cgc acc gtg ccg ttc gtc tccaag 4082 Glu Ala Asn Pro Arg Ala Ser Arg Thr Val Pro Phe Val Ser Lys1250 1255 1260 gca acg ggc gtc aac ctg gcc aag gca gca tcc cgt atc gcagtg 4127 Ala Thr Gly Val Asn Leu Ala Lys Ala Ala Ser Arg Ile Ala Val1265 1270 1275 ggc gcc acc atc aag gat ctc caa gat gag ggc atg att cctacc 4172 Gly Ala Thr Ile Lys Asp Leu Gln Asp Glu Gly Met Ile Pro Thr1280 1285 1290 gag tac gac ggc ggc tcc ttg cca ctg gac gct cca atc gctgtg 4217 Glu Tyr Asp Gly Gly Ser Leu Pro Leu Asp Ala Pro Ile Ala Val1295 1300 1305 aag gaa gca gtg ttg ccg ttc aac cgc ttc cgt cgc cca gatgga 4262 Lys Glu Ala Val Leu Pro Phe Asn Arg Phe Arg Arg Pro Asp Gly1310 1315 1320 aag acc ctg gac acc ctg ctt tcc cca gag atg aag tcc actggc 4307 Lys Thr Leu Asp Thr Leu Leu Ser Pro Glu Met Lys Ser Thr Gly1325 1330 1335 gag gtc atg ggc ttg gcc aac aac ttc ggc gct gca tat gcaaag 4352 Glu Val Met Gly Leu Ala Asn Asn Phe Gly Ala Ala Tyr Ala Lys1340 1345 1350 gct gaa gct ggc gcg ttt ggt gca ttg cca acc gaa ggc accgtc 4397 Ala Glu Ala Gly Ala Phe Gly Ala Leu Pro Thr Glu Gly Thr Val1355 1360 1365 ttc gtg acc gtg gct aac cgc gac aag cgc acc ctg atc ctgcca 4442 Phe Val Thr Val Ala Asn Arg Asp Lys Arg Thr Leu Ile Leu Pro1370 1375 1380 atc cag cgc ctg gcg tcg atg ggc tac aag atc ctc gcc accgaa 4487 Ile Gln Arg Leu Ala Ser Met Gly Tyr Lys Ile Leu Ala Thr Glu1385 1390 1395 ggc acc gca ggc atg ctg cgc cgc aac ggc att gat tgt gaagtt 4532 Gly Thr Ala Gly Met Leu Arg Arg Asn Gly Ile Asp Cys Glu Val1400 1405 1410 gtg ctc aag gct tcc gac atc cgc gaa ggt gta gag ggc aagtcc 4577 Val Leu Lys Ala Ser Asp Ile Arg Glu Gly Val Glu Gly Lys Ser1415 1420 1425 atc gtg gat cgt atc cgc gaa ggc gaa gtt gac ctc atc ctcaac 4622 Ile Val Asp Arg Ile Arg Glu Gly Glu Val Asp Leu Ile Leu Asn1430 1435 1440 acc cca gct ggt tct gct ggc gct cgc cac gat ggc tac gatatc 4667 Thr Pro Ala Gly Ser Ala Gly Ala Arg His Asp Gly Tyr Asp Ile1445 1450 1455 cgc gca gca gca gtg acc gtg ggt gtt cca ctg atc acc actgtc 4712 Arg Ala Ala Ala Val Thr Val Gly Val Pro Leu Ile Thr Thr Val1460 1465 1470 cag ggt gtc acc gca gct gtc cag ggc att gag gcc ctg cgtgag 4757 Gln Gly Val Thr Ala Ala Val Gln Gly Ile Glu Ala Leu Arg Glu1475 1480 1485 ggc gtt gtc agc gtc cgc gcg ctg cag gaa ctc gac cac gcagtc 4802 Gly Val Val Ser Val Arg Ala Leu Gln Glu Leu Asp His Ala Val1490 1495 1500 aag gct taagccctat gacattcggc gagaagctt 4837 Lys Ala 15052 393 PRT Brevibacterium lactofermentum 2 Val Ser Lys Asp Thr Thr ThrTyr Gln Gly Val Thr Glu Ile Gly Ser 1 5 10 15 Val Pro Ala Tyr Leu ValLeu Ala Asp Gly Arg Thr Phe Thr Gly Phe 20 25 30 Gly Phe Gly Ala Ile GlyThr Thr Leu Gly Glu Ala Val Phe Thr Thr 35 40 45 Ala Met Thr Gly Tyr GlnGlu Thr Met Thr Asp Pro Ser Tyr His Arg 50 55 60 Gln Ile Val Val Ala ThrAla Pro Gln Ile Gly Asn Thr Gly Trp Asn 65 70 75 80 Asp Glu Asp Asn GluSer Arg Asp Gly Lys Ile Trp Val Ala Gly Leu 85 90 95 Val Ile Arg Asp LeuAla Ala Arg Val Ser Asn Trp Arg Ala Thr Thr 100 105 110 Ser Leu Gln GlnGlu Met Ala Asp Gln Gly Ile Val Gly Ile Gly Gly 115 120 125 Ile Asp ThrArg Ala Leu Val Arg His Leu Arg Asn Glu Gly Ser Ile 130 135 140 Ala AlaGly Ile Phe Ser Gly Ala Asp Ala Gln Arg Pro Val Glu Glu 145 150 155 160Leu Val Glu Ile Val Lys Asn Gln Pro Ala Met Thr Gly Ala Asn Leu 165 170175 Ser Val Glu Val Ser Ala Asp Glu Thr Tyr Val Ile Glu Ala Glu Gly 180185 190 Glu Glu Arg His Thr Val Val Ala Tyr Asp Leu Gly Ile Lys Gln Asn195 200 205 Thr Pro Arg Arg Phe Ser Ala Arg Gly Val Arg Thr Val Ile ValPro 210 215 220 Ala Glu Thr Pro Leu Glu Asp Ile Lys Gln Tyr Asn Pro SerGly Val 225 230 235 240 Phe Ile Ser Asn Gly Pro Gly Asp Pro Ala Ala AlaAsp Val Met Val 245 250 255 Asp Ile Val Arg Glu Val Leu Glu Ala Asp IlePro Phe Phe Gly Ile 260 265 270 Cys Phe Gly Asn Gln Ile Leu Gly Arg AlaPhe Gly Met Glu Thr Tyr 275 280 285 Lys Leu Lys Phe Gly His Arg Gly IleAsn Val Pro Val Lys Asn His 290 295 300 Ile Thr Gly Lys Ile Asp Ile ThrAla Gln Asn His Gly Phe Ala Leu 305 310 315 320 Lys Gly Glu Ala Gly GlnGlu Phe Glu Thr Asp Phe Gly Thr Ala Ile 325 330 335 Val Thr His Thr CysLeu Asn Asp Gly Val Val Glu Gly Val Ala Leu 340 345 350 Lys Ser Gly ArgAla Tyr Ser Val Gln Tyr His Pro Glu Ala Ala Ala 355 360 365 Gly Pro AsnAsp Ala Ser Pro Leu Phe Asp Gln Phe Val Glu Leu Met 370 375 380 Asp AlaAsp Ala Gln Lys Lys Gly Ala 385 390 3 1113 PRT Brevibacteriumlactofermentum 3 Met Pro Lys Arg Ser Asp Ile Asn His Val Leu Val Ile GlySer Gly 1 5 10 15 Pro Ile Val Ile Gly Gln Ala Cys Glu Phe Asp Tyr SerGly Thr Gln 20 25 30 Ala Cys Arg Val Leu Lys Glu Glu Gly Leu Arg Val ThrLeu Ile Asn 35 40 45 Ser Asn Pro Ala Thr Ile Met Thr Asp Pro Glu Met AlaAsp His Thr 50 55 60 Tyr Val Glu Pro Ile Glu Pro Glu Tyr Ile Asp Lys IlePhe Ala Lys 65 70 75 80 Glu Ile Glu Gln Gly His Pro Ile Asp Ala Val LeuAla Thr Leu Gly 85 90 95 Gly Gln Thr Ala Leu Asn Ala Ala Ile Gln Leu AspArg Leu Gly Ile 100 105 110 Leu Glu Lys Tyr Gly Val Glu Leu Ile Gly AlaAsp Ile Asp Ala Ile 115 120 125 Glu Arg Gly Glu Asp Arg Gln Lys Phe LysAsp Ile Val Thr Thr Ile 130 135 140 Gly Gly Glu Ser Ala Arg Ser Arg ValCys His Asn Met Asp Glu Val 145 150 155 160 His Glu Thr Val Ala Glu LeuGly Leu Pro Val Val Val Arg Pro Ser 165 170 175 Phe Thr Met Gly Gly LeuGly Ser Gly Leu Ala Tyr Asn Thr Glu Asp 180 185 190 Leu Glu Arg Ile AlaGly Gly Gly Leu Ala Ala Ser Pro Glu Ala Asn 195 200 205 Val Leu Ile GluGlu Ser Ile Leu Gly Trp Lys Glu Phe Glu Leu Glu 210 215 220 Leu Met ArgAsp Thr Ala Asp Asn Val Val Val Ile Cys Ser Ile Glu 225 230 235 240 AsnVal Asp Ala Leu Gly Val His Thr Gly Asp Ser Val Thr Val Ala 245 250 255Pro Ala Leu Thr Leu Thr Asp Arg Glu Phe Gln Lys Met Arg Asp Gln 260 265270 Gly Ile Ala Ile Ile Arg Glu Val Gly Val Asp Thr Gly Gly Cys Asn 275280 285 Ile Gln Phe Ala Ile Asn Pro Val Asp Gly Arg Ile Ile Thr Ile Glu290 295 300 Met Asn Pro Arg Val Ser Arg Ser Ser Ala Leu Ala Ser Lys AlaThr 305 310 315 320 Gly Phe Pro Ile Ala Lys Met Ala Ala Lys Leu Ala IleGly Tyr Thr 325 330 335 Leu Asp Glu Ile Thr Asn Asp Ile Thr Gly Glu ThrPro Ala Ala Phe 340 345 350 Glu Pro Thr Ile Asp Tyr Val Val Val Lys AlaPro Arg Phe Ala Phe 355 360 365 Glu Lys Phe Val Gly Ala Asp Asp Thr LeuThr Thr Thr Met Lys Ser 370 375 380 Val Gly Glu Val Met Ser Leu Gly ArgAsn Tyr Ile Ala Ala Leu Asn 385 390 395 400 Lys Ala Leu Arg Ser Leu GluThr Lys Gln Gln Gly Phe Trp Thr Lys 405 410 415 Pro Asp Glu Phe Phe AlaGly Glu Arg Ala Thr Asp Lys Ala Ala Val 420 425 430 Leu Glu Asp Leu LysArg Pro Thr Glu Gly Arg Leu Tyr Asp Val Glu 435 440 445 Leu Ala Met ArgLeu Gly Ala Ser Val Glu Glu Leu Tyr Glu Ala Ser 450 455 460 Ser Ile AspPro Trp Phe Leu Ala Glu Leu Glu Ala Leu Val Gln Phe 465 470 475 480 ArgGln Lys Leu Val Asp Ala Pro Phe Leu Asn Glu Asp Leu Leu Arg 485 490 495Glu Ala Lys Phe Met Gly Leu Ser Asp Leu Gln Ile Ala Ala Leu Arg 500 505510 Pro Glu Phe Ala Gly Glu Asp Gly Val Arg Thr Leu Arg Leu Ser Leu 515520 525 Gly Ile Arg Pro Val Phe Lys Thr Val Asp Thr Cys Ala Ala Glu Phe530 535 540 Glu Ala Lys Thr Pro Tyr His Tyr Ser Ala Tyr Glu Leu Asp ProAla 545 550 555 560 Ala Glu Ser Glu Val Ala Pro Gln Thr Glu Arg Glu LysVal Leu Ile 565 570 575 Leu Gly Ser Gly Pro Asn Arg Ile Gly Gln Gly IleGlu Phe Asp Tyr 580 585 590 Ser Cys Val His Ala Ala Leu Glu Leu Ser ArgVal Gly Tyr Glu Thr 595 600 605 Val Met Val Asn Cys Asn Pro Glu Thr ValSer Thr Asp Tyr Asp Thr 610 615 620 Ala Asp Arg Leu Tyr Phe Glu Pro LeuThr Phe Glu Asp Val Met Glu 625 630 635 640 Val Tyr His Ala Glu Ala GlnSer Gly Thr Val Ala Gly Val Ile Val 645 650 655 Gln Leu Gly Gly Gln ThrPro Leu Gly Leu Ala Asp Arg Leu Lys Lys 660 665 670 Ala Gly Val Pro ValIle Gly Thr Ser Pro Glu Ala Ile Asp Met Ala 675 680 685 Glu Asp Arg GlyGlu Phe Gly Ala Leu Leu Asn Arg Glu Gln Leu Pro 690 695 700 Ala Pro AlaPhe Gly Thr Ala Thr Ser Phe Glu Glu Ala Arg Thr Val 705 710 715 720 AlaAsp Glu Ile Ser Tyr Pro Val Leu Val Arg Pro Ser Tyr Val Leu 725 730 735Gly Gly Arg Gly Met Glu Ile Val Tyr Asp Glu Ala Ser Leu Glu Asp 740 745750 Tyr Ile Asn Arg Ala Thr Glu Leu Ser Ser Asp His Pro Val Leu Val 755760 765 Asp Arg Phe Leu Asp Asn Ala Ile Glu Ile Asp Val Asp Ala Leu Cys770 775 780 Asp Gly Asp Glu Val Tyr Leu Ala Gly Val Met Glu His Ile GluGlu 785 790 795 800 Ala Gly Ile His Ser Gly Asp Ser Ala Cys Ala Leu ProPro Met Thr 805 810 815 Leu Gly Ala Gln Asp Ile Glu Lys Val Arg Glu AlaThr Lys Lys Leu 820 825 830 Ala Leu Gly Ile Gly Val Gln Gly Leu Met AsnVal Gln Tyr Ala Leu 835 840 845 Lys Asp Asp Ile Leu Tyr Val Ile Glu AlaAsn Pro Arg Ala Ser Arg 850 855 860 Thr Val Pro Phe Val Ser Lys Ala ThrGly Val Asn Leu Ala Lys Ala 865 870 875 880 Ala Ser Arg Ile Ala Val GlyAla Thr Ile Lys Asp Leu Gln Asp Glu 885 890 895 Gly Met Ile Pro Thr GluTyr Asp Gly Gly Ser Leu Pro Leu Asp Ala 900 905 910 Pro Ile Ala Val LysGlu Ala Val Leu Pro Phe Asn Arg Phe Arg Arg 915 920 925 Pro Asp Gly LysThr Leu Asp Thr Leu Leu Ser Pro Glu Met Lys Ser 930 935 940 Thr Gly GluVal Met Gly Leu Ala Asn Asn Phe Gly Ala Ala Tyr Ala 945 950 955 960 LysAla Glu Ala Gly Ala Phe Gly Ala Leu Pro Thr Glu Gly Thr Val 965 970 975Phe Val Thr Val Ala Asn Arg Asp Lys Arg Thr Leu Ile Leu Pro Ile 980 985990 Gln Arg Leu Ala Ser Met Gly Tyr Lys Ile Leu Ala Thr Glu Gly Thr 9951000 1005 Ala Gly Met Leu Arg Arg Asn Gly Ile Asp Cys Glu Val Val Leu1010 1015 1020 Lys Ala Ser Asp Ile Arg Glu Gly Val Glu Gly Lys Ser IleVal 1025 1030 1035 Asp Arg Ile Arg Glu Gly Glu Val Asp Leu Ile Leu AsnThr Pro 1040 1045 1050 Ala Gly Ser Ala Gly Ala Arg His Asp Gly Tyr AspIle Arg Ala 1055 1060 1065 Ala Ala Val Thr Val Gly Val Pro Leu Ile ThrThr Val Gln Gly 1070 1075 1080 Val Thr Ala Ala Val Gln Gly Ile Glu AlaLeu Arg Glu Gly Val 1085 1090 1095 Val Ser Val Arg Ala Leu Gln Glu LeuAsp His Ala Val Lys Ala 1100 1105 1110 4 32 DNA Artificial Sequencesynthetic DNA 4 cccgttaact gcttgaaacc caggacaata ac 32 5 30 DNAArtificial Sequence synthetic DNA 5 cccgttaaca tgtacttcag aaaagattag 306 26 DNA Artificial Sequence synthetic DNA 6 gatatctacg tgccgatcaacgtctc 26 7 25 DNA Artificial Sequence synthetic DNA 7 aggcctttttttaaggcagt tattg 25

What is claimed is:
 1. A DNA fragment which encodes a polypeptidedefined in the following (A) or (B): (A) a polypeptide which has anamino acid sequence comprises at least the amino acid numbers 50 to 393of the amino acid sequence of SEQ ID NO: 2, (B) a polypeptide which hasan amino acid sequence comprises at least the amino acid numbers 50 to393 of the amino acid sequence of SEQ ID NO: 2 including substitution,deletion, insertion, addition, or inversion of one or several aminoacids, and can constitute a protein having a carbamoyl-phosphatesynthetase activity with a large subunit of carbamoyl-phosphatesynthetase comprising the amino acid sequence of SEQ ID NO:
 3. 2. A DNAfragment which encodes a polypeptide defined in the following (C) or(D): (C) a polypeptide which comprises the amino acid sequence of SEQ IDNO: 3, (D) a polypeptide which comprises the amino acid sequence of SEQID NO: 3 including substitution, deletion, insertion, addition, orinversion of one or several amino acids, and can constitute a proteinhaving a carbamoyl-phosphate synthetase activity with a small subunit ofcarbamoyl-phosphate synthetase having an amino acid sequence comprisesat least the amino acid numbers 50 to 393 of the amino acid sequence ofSEQ ID NO:
 2. 3. A DNA fragment encoding a polypeptide which comprisesthe amino acid sequence of SEQ ID NO: 3 including substitution,deletion, insertion, addition, or inversion of one or several aminoacids, and can constitute a protein having a carbamoyl-phosphatesynthetase activity.
 4. A DNA fragment which encodes a polypeptidedefined in the following (a) or (b), and a polypeptide defined in thefollowing (c) or (d): (a) a polypeptide which has an amino acid sequencecomprising at least the amino acid numbers 50 to 393 in SEQ ID NO: 2,(b) a polypeptide which has an amino acid sequence comprising at leastthe amino acid numbers 50 to 393 in SEQ ID NO: 2 including substitution,deletion, insertion, addition, or inversion of one or several aminoacids, and can constitute a protein having a carbamoyl-phosphatesynthetase activity with a large subunit of carbamoyl-phosphatesynthetase comprising the amino acid sequence of SEQ ID NO: 3, (c) apolypeptide which comprises the amino acid sequence of SEQ ID NO: 3, (d)a polypeptide which comprises the amino acid sequence of SEQ ID NO: 3including substitution, deletion, insertion, addition, or inversion ofone or several amino acids, and can constitute a protein having acarbamoyl-phosphate synthetase activity with a small subunit ofcarbamoyl-phosphate synthetase having an amino acid sequence comprisingthe amino acid numbers 50 to 393 in SEQ ID NO:
 2. 5. The DNA fragmentaccording to claim 1, which has a nucleotide sequence comprising atleast the nucleotide numbers 430 to 1461 in the nucleotide sequence ofSEQ ID NO:
 1. 6. The DNA fragment according to claim 2, which has anucleotide sequence comprising at least the nucleotide numbers 1756 to4809 in the nucleotide sequence of SEQ ID NO:
 1. 7. The DNA fragmentaccording to claim 3, which has a nucleotide sequence comprising atleast the nucleotide numbers 430 to 4809 in the nucleotide sequence ofSEQ ID NO:
 1. 8. A protein which comprises a polypeptide defined in thefollowing (a) or (b), and a polypeptide defined in the following (c) or(d): (a) a polypeptide which has an amino acid sequence comprising atleast the amino acid numbers 50 to 393 in SEQ ID NO: 2, (b) apolypeptide which has an amino acid sequence comprising at least theamino acid numbers 50 to 393 in SEQ ID NO: 2 including substitution,deletion, insertion, addition, or inversion of one or several aminoacids, and can constitute a protein having a carbamoyl-phosphatesynthetase activity with a large subunit of carbamoyl-phosphatesynthetase comprising the amino acid sequence of SEQ ID NO: 3, (c) apolypeptide which comprises the amino acid sequence of SEQ ID NO: 3, (d)a polypeptide which comprises the amino acid sequence of SEQ ID NO: 3including substitution, deletion, insertion, addition, or inversion ofone or several amino acids, and can constitute a protein having acarbamoyl-phosphate synthetase activity with a small subunit ofcarbamoyl-phosphate synthetase having an amino acid sequence comprisingat least the amino acid numbers 50 to 393 in SEQ ID NO:
 2. 9. Acoryneform bacterium which is transformed with a DNA fragment accordingto any one of claims 1 to
 7. 10. A microorganism which has enhancedintracellular carbamoyl-phosphate synthetase activity, and hasL-arginine productivity.
 11. The microorganism according to claim 10,wherein the enhanced intracellular carbamoyl-phosphate synthetaseactivity is obtained by increasing copy number of DNA encodingcarbamoyl-phosphate synthetase of the microorganism, or by modifying anexpression regulation sequence so that expression of the gene encodingcarbamoyl-phosphate synthetase in the cell should be enhanced.
 12. Themicroorganism according to claim 11, wherein the DNA is a DNA fragmentaccording to any one of claims 1 to
 7. 13. The microorganism accordingto claim 12, which is a coryneform bacterium.
 14. A method for producingof L-arginine, comprising the steps of culturing a coryneform bacteriumaccording to any one of claims 10 to 13 in a medium to produce andaccumulate L-arginine in the medium, and collecting the L-arginine fromthe medium.